Introduction: Availability of a rapid and reliable platelet activation assay avoiding limitations of current techniques would be valuable to diagnose heparin-induced thrombocytopenia and platelet secretion disorders. Objectives: The first aim was to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify in a single run TxB2 synthesized and serotonin released from platelets. The second aim was to use our method in association with light transmission aggregometry (LTA) to select good platelet responders for the diagnosis of HIT. Methods: Electrospray ionization and chromatographic separation were optimized for the simultaneous dosage of serotonin and TxB2. The method was validated according to the European Medicines Agency (EMA) guideline for bioanalytical method validation. LTA was performed with monoclonal anti-CD9 (clone ALB6) as platelet activator to select good responders. Results: Detection was performed using a tandem mass spectrometer with alternated positive and negative electrospray ionization. The total run time was 6 minutes. The method was validated for calibration curves, precision, accuracy, lower limit of quantification, carry-over, selectivity, and matrix effect. Platelet response to ALB6 was highly variable among donors. Conclusion: We developed and validated a UHPLC-MS/MS method for the simultaneous quantification of TxB2 and serotonin.